ISOLATION OF NORMAL HUMAN COLONIC MUCOSA: COMPARISON OF METHODS

摘要

The commonly used methods for isolation and suspension culture of human colonic epithelium from specimens of colonic mucosae may be characterized as those using mainly mechanical dissociation (8,14,15), and those using chelation with minimal mechanical agitation (6,20). To date, routine long-term culture of normal colonic-epithelial cells on a surface substratum has not been developed, although short-term cultures (less than 7 d) of this type have been reported using methodology employing mechanicaldissociation with enzymatic digestion (5). Authors reporting viable long-term suspension cultures (3-5 mo.) of human colonic epithelial cells (14) have not demonstrated retention of normal crypt architecture nor tried to remove stromal components. However, methods for isolating human colonic crypts/epithelial cells that eliminate stromal components from tissue used to initiate cultures apparently lead only to short-term cultures (5,8,20). Therefore, we decided to compare colonic epithelium isolated mechanically by Moyer's procedure (14) to that obtained by the primarily chelation-based (nonenzymatic) method of Whitehead et al. (20) on the basis of ultrastructural morphology, architectural integrity of crypts, and degree of stromal contamination. In addition, we determined whether crypts isolated by the chelation-based method could yield short-term outgrowths similar to those reported by Buset et al. (5), who used mechanical isolation plus enzymatic digestion.